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We describe the reconstitution of dynamic cortical actin networks within cell-like compartments. The approach is based on encapsulation of Xenopus egg extract within water-in-oil emulsions. The growth of cortical actin networks is catalyzed by an amphiphilic actin nucleation-promoting factor that localizes to the water/oil interface. We first describe the preparation of cell-free Xenopus egg extract that provides all the soluble components of the actin machinery. We then describe the preparation of the amphiphilic, fluorescent actin nucleation-promoting factor that directs actin polymerization to the interface. Finally, we describe the steps required for assembly of dynamic actin cortices within water-in-oil emulsions, including the emulsification process and the sample preparation procedures. We provide recommendations for handling sensitive system components and discuss potential uses of this reconstitution approach for cytoskeletal research.

Original publication

DOI

10.1016/bs.mcb.2015.01.011

Type

Journal article

Journal

Methods Cell Biol

Publication Date

2015

Volume

128

Pages

287 - 301

Keywords

Actin, Artificial cells, Cortex, Emulsions, In vitro reconstitution, Actin Cytoskeleton, Actins, Animals, Cell-Free System, Emulsions, Mineral Oil, Ovum, Tissue Extracts, Trimethylsilyl Compounds, Water, Xenopus